Distinct [Ca²⁺]_i elevations in platelets interacting with acid-soluble type I collagen under flow. Washed human platelets (2 × 10⁷/mL) loaded with 8 μM FLUO 3-AM were suspended with erythrocytes (40% hematocrit) in HEPES–Tyrode buffer containing 2 mM Ca²⁺ and Mg²⁺ and perfused over collagen for 10 minutes at the shear rate of 250 seconds. The low platelet count was required for accurate image analysis. (A) After perfusion for 120 seconds, [Ca²⁺]_i in surface-interacting platelets was monitored for 90 seconds. Both short-lasting (α-like) and long-lasting (γ-like) peaks were observed. (B) An anti-αIIbβ3 antibody (LJ-CP8, 100 μg/mL) had no effect on Ca²⁺ signals. (C) Platelets treated with an anti-α2β1 antibody (R2-7E4, 100 μg/mL) exhibited no [Ca²⁺]_i oscillations and only transient contacts with the surface. (D) An anti-GPVI antibody (Fab 9O12.2, 50 μg/mL) had no apparent effect on α-like but abolished γ-like [Ca²⁺]_i oscillations. (E) The number of activated platelets exhibiting at least one α-like (■) or γ-like () [Ca²⁺]_i elevation over a 3-minute period was measured as a fraction of the total number of surface interacting platelets, in the absence (control) or presence of different antibodies, as indicated. (F) The number of α-like [Ca²⁺]_i elevations in each activated platelet was measured over a 3-minute period, in the absence (control) or presence of different monoclonal antibodies, as indicated. At least 250 control or antibody-treated platelets were evaluated. (G) Number of control and antibody-treated platelets (including transient and stable adhesion) present in each optical field measured every 10 seconds for 200 seconds. The results shown in panels B to D represent the mean ± 95% CI of the values measured in at least 8 experiments. Significant difference from the corresponding control: *P < .05; **P < .01; ***P < .001.